This invention relates to two pure strains of bacteria capable of selective removal of organically bound sulfur from carbonaceous materials while maintaining the calorific value of the carbonaceous materials. The microorganisms of this invention are particularly useful in removal of organic sulfur from fossil fuels such as oils and coals.
Conventional methods for the removal of organic sulfur from fuels rely on heterogeneous catalysis which is limited by the range of compounds which can be successfully removed. Aromatic sulfur containing compounds make up a significant percentage of the organic sulfur in fuels and these are generally the least susceptible to removal by conventional methods. The most recalcitrant of the aromatic sulfur-containing compounds are those in which the sulfur atoms are sterically hindered.
Most microbial methods described to date for the removal of sulfur from petroleum products, coal, tar sands and shale oil are limited to inorganic sulfur. The majority of the microbial methods involving organic sulfur removal are non-specific in that they attack the carbon skeletons of the organic constituents, thus resulting in a high degree of unwanted side reactions and reduction of the calorific value of the fuel. In addition, there have been no reports on the selective removal of sulfur from those organic constituents in which the sulfur atoms are sterically hindered.
The following is a brief description of the prior art.
Sulfur in carbonaceous fuels is undesirable due to its ability to promote corrosion in storage and processing equipment and, when the fuel is combusted, the release of sulfur oxide gases which are attributed to detrimental affects on the environment. Sulfur is found in two forms in these materials: 1) inorganic, with the principle forms being elemental sulfur, sulfate, and pyrite; and 2) organic sulfur, with the principle forms in crude oils being (Rall, H. T., Thompson, C. J., Coleman, H. J., and Hopkins, R. L. (1972) Sulfur compounds in crude oil. U.S. Department of the Interior, Bureau of Mines, Bulletin 659) aliphatic and aromatic thiols, dialkyl, diaryl, alkyl-aryl and cyclic sulfides, disulfides, and thiophenes such as thiophene, benzothiophene, dibenzothiophene and various derivatives of these compounds to which alkyl and/or aryl groups are attached. Although the structure of coal is not known the sulfur forms are believed to be similar to oils except that they are covalently bound in the complex coal matrix.
Coal can contain appreciable quantities of both forms of sulfur with a total sulfur content, typically between 1 and 4%, but in some cases being greater than 11% (Chakrabarti, J. N. (1978) Analytical Methods for Coal and Coal Products, 1,279-322. New York: Academic). The sulfur content of crude oils varies widely ranging from less than 0.1% to 5% and more in some of the heavy crudes (International Petroleum Encyclopedia. (1983) Penn Well Publishing Co., Tulsa, Okla.). In crude oils sulfur is also found in both inorganic and organic forms, in addition, hydrogen sulfide may also be present. In distillate fractions of crude oils the sulfur content increases with the boiling range of the fraction with the bulk of the sulfur found in the middle distillates fractions and above.
A number of processes have been developed to remove inorganic sulfur forms from coal including oxidation to sulfur oxide gases and reduction to hydrogen sulfide gas, and physical cleaning by froth flotation and upward current classifiers based on the substantial difference in density of coal and pyrite. In addition, a number of microbial approaches have been developed which rely largely on the oxidation of pyrite and reduced sulfur forms, including elemental sulfur, by Thiobacillus specia and Sulfolobus species, releasing sulfur in the form of sulfuric acid and sulfates (reviewed in: Bos, P., and Kuenen, J. G. (1990) Microbial treatment of coal. In: Microbial Mineral Recovery (Ehrlich, H. L., and Briedey, C. L. eds) McGraw Hill, New York, pp. 343-377). Some of the microbial process have been patented, exemplified by Detz, C. M., and Barvinchak, G. (1978) U.S. Pat. No. 4,206,288 in which Thiobacillus ferroxidans is employed in a slurry reactor design to remove inorganic sulfur from coal, and Attia, Y. A. and Elzeky, M. A. (1988) U.S. Pat. No. 4,775,627 in which Thiobacillus ferroxidans is used in conjunction with physical separation to remove inorganic sulfur.
The removal of organic sulfur is more problematic. In coal organic sulfur is covalently bound within the complex carbonaceous structure. For precombustion sulfur removal this requires that the coal matrix be exposed to the desulfurizing agent by depolymerization and/or solubilized prior to treatment. Once pretreated in this way the organic sulfur in the coal product is removed by similar techniques as those used for oils. The requirement for pretreatment is a major factor in determining costs for a coal desulfurization process. Alternatively, organic sulfur in coal is removed by scrubbing of sulfur dioxide after combustion. The use of scrubbers typically limits the use of coal to large facilities were their use is economically feasible (Bos, P., and Kuenen, J. G. (1990) Microbial treatment of coal. In: Microbial Mineral Recovery (Ehrlich, H. L., and Brierley, C. L., eds) McGraw-Hill, New York, pp. 343-377).
In standard refinery operations organic sulfur in oils is removed by hydrodesulfurization using heterogeneous inorganic catalysts, high temperature and high hydrogen pressure. These methods are effective in removing thiols, most sulfides and disulfides but are much less effective against thiophenic sulfur, particularly the dibenzothiophenes containing substitutions rendering the sulfur atom sterically hindered, i.e., monobeta and dibeta substituted dibenzothiophenes. Tighter regulations on the maximum mount of sulfur allowable in fuels will necessitate the removal of even the most recalcitrant sulfur compounds. This requires the development of new technologies or the processing of fuels under much more severe conditions, i.e., increased temperature and hydrogen pressure, thereby greatly increasing the cost of fuel processing.
A large amount of research and many patents have been issued on the use of microorganisms to remove organic sulfur from coal and oils (reviewed in: 1) Bos, P. and Kuenen, J. G. (1990) Microbial treatment of coal. In: Microbial Mineral Recovery (Ehrlich, H. L., and Brierlcy, C. L., eds) McGraw-Hill, New York, pp. 343-377:2) Foght, J. M., Fedorak, P. M., Gray, M. R., and Westlake, D. W. S. (1990) Microbial desulfurization of petroleum. In: Microbial Mineral Recovery (Ehrlich, H. L., and Brierley, C. L., eds) McGraw-Hill, New York, pp. 379-407). The majority of this work has focused on aerobic processes using dibenzothiophene as the model compound to isolate organisms and evaluate reaction mechanisms. Research on the use of anaerobic bacteria to remove sulfur via a reductive process resulting in the release of sulfur as hydrogen sulfide is exemplified by Kim, B. H., Kim, T. S., and Kin, H. Y. (1990) U.S. Pat. No. 4,954,229, in which electrical energy is used to supply reducing power for the reduction or organic sulfur by Desulfovibrio species.
The vast majority of microorganisms isolated which can degrade dibenzothiophene aerobically do so by the initial oxidation and cleavage of one of the aromatic rings, and in so doing initiate the complete degradation of the molecule to CO.sub.2, H.sub.2 O and SO.sub.4 .dbd.. This pathway was initially described by Kodama, et al. (Kodama, Kl., Nakatani, S., Umehara, K., Shimizu, K., Minoda, Y., and Yamada, K. (1970) Microbial conversion of petrosulfur compounds: Part III. Isolation and identification of products from dibenzothiophene. Agr. Biol. Chem. 34, 1320-1324) and is frequently referred to as the Kodama pathway.
Microbial degradation of organosulfur containing carbonaceous materials by the Kodama pathway, or related pathways involving C--C bond cleavage, is undesirable due to the lack of specificity for sulfur. These nonspecific pathways result in the degradation of structurally related aromatic hydrocarbon compounds thereby greatly reducing the efficiency of the process and the calorific value of the fuel. The lack of sulfur specificity inherent in Kodama like pathways is illustrated by the work of Monticello, et al. (Monticello, D. J., Bakker, D., Schell, M., and Finnerty, W. R. (1985) Appl. Environ. Microbiol. 49, 761-764) who, working with Pseudomonas species, demonstrated that mutants unable to grow on dibenzothiophene were also unable to oxidize naphthalene. It is therefore desirable to utilize a microbial desulfurization process which removes organically bound sulfur via a sulfur specific mechanism without removing carbon from the molecule, thereby operating at an efficiency, and retaining the calorific value of the fuel, in a manner not possible by carbon degradative pathways.
Sulfur specific oxidation of dibenzothiophene by ATCC 39381 resulting in the release of sulfur as sulfate without the degradation of the carbon skeleton is described by Isbister and Doyle (Isbister, J. S., and Doyle, R. C. (Atlantic Res. Corp.) (1985) U.S. Pat. No. 4,562,156). However, the ATCC 39381 culture on deposit does not possess the C-S cleavage trait and the depositors of the culture have stated that the culture on deposit cannot be replaced as such cultures having the C-S cleavage trait to their knowledge do not exist (4th Department of Energy Preparation, Utilization and Environmental Control Contractors Conference, U.S. Department of Energy, Pittsburgh Energy Technology Center, Pittsburgh, Pa. 15236, U.S.A., 1988). More recently, a patent by Kilbane (Kilbane, J. J. (1991) U.S. Pat. No. 5,002,888) describes the use of a mutant strain of Bacillus sphaericus strain ATCC 53969 which has the property of sulfur removal from organosulfur compounds by selective cleavage of C--S bonds. In the case of sulfur specific metabolism of dibenzothiophene, the end products are 2-hydroxy bi-phenyl and SO.sub.4 .dbd.. This organism is unable to perform the desulfurization of organosulfur compounds independently, requiring the presence of a "nutritional helper culture". Kilbane and Bielalga have reported the isolation of a mutant strain of Rhodococcus rhodochrous strain IGTS8 which performs the identical sulfur specific metabolism of organosulfur compounds but does not require a "helper culture" (Kilbane, J. J. and Bielaga, B. A. (1990) Microbial removal or organic sulfur from coal: a molecular genetics approach. In: Gas, Oil, Coal, and Environmental biotechnology II (Akin, C. and Smith, J. eds.) Institute for gas technology, Chicago, pp. 317-330) van Afferden, et al. (van Afterden, M., Schacht, S., Klein, J. and Truper, H. G. Degradation of dibenzothiophene by Brevibacterium sp. Do. Arch. Microbiol. 153,324-328) reported the isolation of a Brevibacterium species which is able to use dibenzothiophene as a sole carbon, sulfur and energy sources and therefore completely degrades the molecule to CO.sub.2 and H.sub.2 O, this process is initiated by oxidation of the sulfur to dibenzothiophene sulfoxide and then to the sulfone, two intermediates in the proposed pathway utilized by IGTS8. Omori, et al., (Omori, T., Monna, L., Saiki, Y., and Kodama, T. (1992). Desulfurization of dibenzothiophene by Corynebacterium sp strain SY1. Appl. Environ. Microbiol. 58, 911-915) reported the isolation of Corynebacterium sp strain SY 1 which selectively removed sulfur from dibenzothiophene and a number of other organosulfur compounds. Corynebacterium sp strain SY 1 metabolized dibenzothiophene to dibenzothiophene-5-oxide, dibenzothiophene sulfone and hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls.
In none of the above cases involving sulfur specific metabolism were sterically hindered organosulfur compounds demonstrated to be substrates for sulfur removal. Further, it is evident that the isolation of organisms capable of sulfur specific metabolism of organosulfur compounds of the type found in carbonaceous materials such as coal and oil is not obvious. This is demonstrated by the work of Kilbane and Bielalga (Kilbane, J. J. and Bielaga, B. A. (1990) Microbial removal of organic sulfur from coal: a molecular genetics approach. In: Gas, Oil, Coal, and Environmental biotechnology II)(Akin, C. and Smith, J. eds.) where three strains of Rhodococcus rhodochrous different than the sulfur specific strain Rhodococcus rhodochrous IGTS8 and five other Rhodococcus strains were shown to lack sulfur specific metabolism of organosulfur compounds. This evidence and work in our own laboratory shows that sulfur specific metabolism of organosulfur compounds is strain specific and not a general characteristic of any genera or species of microorganisms.